Transition metal ions are required for biological survival, yet become toxic at high intracellular concentrations. Metal ion homeostasis allows all organisms, from bacteria to humans, to precisely regulate the bioavailability of essential metal ions while eliminating heavy metal pollutants from the cell. Metal-responsive control of gene expression governs metal homeostasis and is mediated by metalloregulatory proteins. Our long-term goal is to understand how the structure of specific metal coordination complexes leads to allosteric regulation of promoter DNA binding and/or transcription activation by these "metal sensor" proteins. The human zinc sensor, metalloregulatory transcription factor-1 (MTF-1), a potent transcriptional activator of genes that mitigate the effects of heavy metal, hypoxic and oxidative stress, and several members of the SmtB/ArsR family, a large class of prokaryotic metalloregulatory repressors, are proposed for study. Our specific aims are to: 1) Elucidate the metal-binding properties and solve the solution structure of a novel carboxyl-terminal cysteine-rich metal-sensing domain of human MTF-1; 2) Test our hypothesis that the N-terminal domain of hMTF-1, proximal to the zinc finger DNA-binding domain, adopts a bromodomain fold that positively influences the ability of MTF-1 to bind chromatin and activate gene expression; 3) Elucidate the metal binding and DNA binding properties of three new SmtB/ArsR metal sensors in an effort to significantly expand our understanding of how this family evolved to functionally discriminate among distinct metal ions; 4) Determine the molecular mechanism of allosteric coupling of metal binding to operator/promoter DNA binding using the Zn/Co sensor S. aureus CzrA and the Ni/Co sensor, M. tuberculosis NmtR as model systems; and 5) Elucidate the kinetics of metal binding by the cadmium sensor S. aureus pl258 CadC vs. CzrA and NmtR using stopped-flow absorbance and fluorescence spectroscopies. Our findings will provide new mechanistic insight into basic mechanisms of transcriptional activation in eukaryotes, metalloprotein evolution, protein design and engineering of new biosensor devices for heavy metal pollutants, and allosteric coupling in macromolecules.